Systems genetics analysis of human body fat distribution genes identifies adipocyte processes

Using Bayesian network analysis, we modeled gene-gene interactions and predicted novel regulators of adipose tissue fat storage. We show that five novel genes affect adipogenesis or mitochondrial function in human fat cells.


Introduction
Obesity (measured as body mass index [BMI]) is a condition that affects roughly 40% of Americans (Hales et al, 2020) and significantly increases the risk of cardio-metabolic disease (Riaz et al, 2018;Cheng et al, 2019).Excess energy is primarily stored as lipids in two large adipose tissue depots, abdominal visceral and lower body subcutaneous.Adipocytes in each depot can expand by increasing their number (through pre-adipocyte proliferation and differentiation) or their size (through adipocyte glucose and free fatty acid uptake, lipogenesis, and lipolysis) to increase storage capacity (Drolet et al, 2008;Haczeyni et al, 2018).Whereas subcutaneous adipocytes expand to store lipids safely and efficiently (Williams et al, 1997), visceral adipocytes are less able to accommodate excess energy through adipocyte expansion (Tchkonia et al, 2005;Pellegrinelli et al, 2016).This causes inflammation (Alvehus et al, 2010;Samaras et al, 2010;Despr és, 2012), insulin resistance (Kabir et al, 2005), hypertension (Mathieu et al, 2009), and negative effects on lipid metabolism (Lemieux et al, 2001) in other tissues.Thus, disproportionate visceral fat storage contributes to the disease risk imparted by over-nutrition.Multiple studies find that the ratio of waist circumference to hip circumference (WHR), an approximation of human fat distribution, and WHR independent of overall obesity (WHR adjBMI ), are both similar or better predictors for cardiovascular disease risk and Type II Diabetes risk than BMI (Schleinitz et al, 2014a;Dale et al, 2017;Emdin et al, 2017).Males generally have higher WHR adjBMI and higher disease risk than females (Williams et al, 1997;Bots et al, 2017;Ye et al, 2019).Our understanding of the genetic and molecular mechanisms that contribute to fat distribution is limited, and thus, unlike overall obesity (Drucker, 2018;Samms et al, 2021), no targeted therapeutics or lifestyle interventions to combat abdominal obesity are known (Kesztyüs et al, 2018).
To date, only five genes (KLF14, LRP5, TBX15, RSPO3, SHOX2) are mechanistically linked to disproportionate fat storage in one depot compared with the other.They affect pathways crucial to the expansion of subcutaneous and visceral adipocyte populations (Gesta et al, 2006(Gesta et al, , 2011;;Lee et al, 2013;Loh et al, 2015Loh et al, , 2020;;Small et al, 2018;Yang et al, 2022).LRP5 and RSPO3 affect adipocyte differentiation by controlling Wnt signaling, whereas TBX15 controls adipocyte differentiation and mitochondrial function.
The Wnt signaling pathway is a well-established driver of cell fate, differentiation, and proliferation in many cell types (Teo & Kahn, 2010); Wnt inhibits adipogenic differentiation by transcriptionally up-regulating osteogenic genes when down-regulating PPARɣ and CEBPα (de Winter & Nusse, 2021).In many contexts, the non-canonical Ca 2+ form of Wnt signaling is inhibitory of the canonical Wnt pathway (Akoumianakis et al, 2022).Wnt signaling activity is positively associated with visceral adiposity (Fuster et al, 2015;Chen & Wang, 2018), and many Wnt pathway genes, especially ligands and receptors used in Ca 2+ non-canonical Wnt signaling, are differentially expressed between fat depots (Zuriaga et al, 2017).
Mitochondrial function correlates strongly with cardiometabolic diseases and can alter adipogenic differentiation (Lee et al, 2019).In mature adipocytes, mitochondria can facilitate physical connections with the lipid droplet (Cui & Liu, 2020), dissipate excess energy via thermogenesis through UCP1 (Montanari et al, 2017), and promote lipid homeostasis (Lee et al, 2019).Human visceral fat has higher mitochondrial activity compared with subcutaneous fat (Pafili et al, 2022), and in multiple metabolic disease states, only visceral mitochondria become dysregulated (Kraunsøe et al, 2010;Pafili et al, 2022).We hypothesize that other putative drivers of fat distribution affect Wnt signaling or mitochondrial function in adipocytes, with different outcomes in each depot.
Many genes potentially contribute to fat distribution.WHR adjBMI is a complex trait that is up to 60% heritable (Schleinitz et al, 2014a), and recent genome-wide association study (GWAS) meta-analyses have uncovered ~350 loci associated with WHR adjBMI .Approximately one-third of the GWAS loci are associated with fat distribution in only one sex (Pulit et al, 2017(Pulit et al, , 2019)), and some have greater magnitude of association in one sex (Winkler et al, 2015).Many of the ~500 genes near these loci have active regulatory regions in adipose tissue (Shungin et al, 2015) and are highly expressed in adipose tissue, many with differential expression between depots (Karastergiou et al, 2013;Schleinitz et al, 2014b).The expression levels of many of these genes are associated with genetic variants within the loci, which are termed expression quantitative trait loci (eQTLs) (Civelek et al, 2017;Raulerson et al, 2019;Wu et al, 2019).Single-gene defects that cause lipodystrophy, an extreme fat distribution phenotype, also affect adipocyte function (Lightbourne & Brown, 2017).
Computational models that describe gene-gene interactions, such as networks, can be powerful tools for interrogating regulatory relationships between many genes at once and prioritizing biologically relevant regulators in the geneset (Schadt & Lum, 2006;Civelek & Lusis, 2014).Correlation-based networks, which identify groups of co-regulated genes (Langfelder & Horvath, 2008), are frequently used to nominate disease regulators, as well as to annotate genes of unknown function or identify conserved gene programs (van Dam et al, 2018).Bayesian networks improve upon correlation-based networks by modeling causal, directed connections, and have been used to understand gene-gene interactions in many human disease models (Zhang et al, 2013(Zhang et al, , 2020;;Hamed et al, 2015;Huan et al, 2015;McKenzie et al, 2017;Shu et al, 2017Shu et al, , 2019;;Agrahari et al, 2018;Chella Krishnan et al, 2018;Carcamo-Orive et al, 2020;Hartman et al, 2021).The directed network structure allows researchers to easily nominate in silico "key drivers" of tissue-specific gene regulation and of disease (Sieberts & Schadt, 2007).Network key driver genes are putatively regulators of tissue-or disease-relevant processes.Whereas this process allows us to prioritize certain GWAS candidate genes over others, it can also nominate candidate regulators that are not genetically regulated.In various cell types and disease models, these key driver genes, both GWAS candidates and other mechanistic genes, are biologically relevant and alter disease-related pathways in vitro (Zhang et al, 2013(Zhang et al, , 2020;;M äkinen et al, 2014;Hamed et al, 2015;Huan et al, 2015;McKenzie et al, 2017;Shu et al, 2017Shu et al, , 2019;;Agrahari et al, 2018;Chella Krishnan et al, 2018;Carcamo-Orive et al, 2020;Hartman et al, 2021).
To identify and prioritize candidate genes involved in fat distribution, we harnessed the predictive power of the Bayesian network construction and key driver analysis approach to identify genes likely to drive fat storage in subcutaneous and visceral fat (Fig 1).Because of the observed differences in body fat distribution between males and females, we constructed separate Bayesian networks of each sex-depot pair to model the distinct gene regulation in each tissue.To increase the predictive power of key driver analysis, we identified key driver genes replicated in two independent cohorts.We used publicly available data to prioritize key driver genes expressed in (pre-)adipocytes, unstudied in adipose tissue, and associated with fat storage in humans or mice.We focused on prioritized key drivers that affect Wnt signaling or mitochondrial function in other cell types, and we functionally profiled seven genes in subcutaneous pre-adipocytes.

Results
Bayesian networks model adipose tissue gene connections in a sex-and depot-specific manner We interrogated two independent datasets of subcutaneous and visceral adipose tissue gene expression, Genotype-Tissue Expression project (GTEx) (The Genotype-Tissue Expression, 2020) and the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) (Franz én et al, 2016) (see the Materials and Methods section) and stratified each dataset by sex.Each dataset has about twice as many males as females (Table 1).
We chose a set of 8,492 genes expressed in these datasets as network inputs, including 495 genes near WHR adjBMI GWAS loci (see the Materials and Methods section, Table S1).We also added prior information about some genes to improve network performance, including genes with eQTLs in the corresponding adipose tissue depot (Table S2).We constructed eight sex-and depot-specific networks (see the Materials and Methods section) that contained an average of 6,250 genes connected, with an average of 6,821 directed edges between those genes (Tables 2 and S3).Each network displayed scale-free and small-world properties consistent with known biological networks (see the Materials and Methods section, Table S4).
Bayesian network structure identifies putative sex-and depotspecific "key drivers" of adipose tissue function and disease Using our model of gene-gene interactions, we identified genes which are regulatory of many others using an approach called key driver analysis (Zhang et al, 2013;Shu et al, 2016).In each network, we identified key driver genes as those that regulated more downstream genes than expected by chance, or those that regulated more downstream genes that were also WHR adjBMI candidate GWAS genes than expected by chance (see the Materials and Methods section, Fig 1).Others find that key drivers genes are more likely than other network features to be reproduced in independent datasets (Cohain et al, 2017) and have been experimentally validated for their regulatory role in cells (Zhang et al, 2013;M äkinen et al, 2014).We identified an average of 691 key driver genes per network (Tables 2 and S5), and we hypothesize that some may regulate fat storage in adipocytes.To avoid problems of overfitting, we compared the key driver genes between the corresponding GTEx and STARNET networks.We identified 334 shared key driver genes in total (Tables 2, S5, and S6).Only 38 shared key driver genes were found in multiple sex-depot groups, and only one key driver, ARHGEF12, was identified in all eight networks.

Overlaps between STARNET and GTEx network key driver genes are significant
We observed that our networks constructed using male data were consistently larger than their female counterparts, with more connected nodes and edges.They had greater predictive power, identifying more genes that were key drivers, and further, the GTEx and STARNET male networks agreed more often, identifying more shared key drivers than female networks.To determine if this overlap between STARNET and GTEx was more than expected by chance, we chose a random set of genes from each network equal to the number of key drivers identified.We compared the shared genes in the random sets 10,000 times to form a null distribution.For the male subcutaneous and male visceral networks, we found that the number of shared key driver genes was significantly greater than random chance (Fig S1A and B).There were more female subcutaneous network key drivers than expected by chance, although the difference was less significant than in male networks, and there were not more female visceral network key drivers than random selection (Fig S1C and D).
To further interrogate the differences in network predictive power because of sex, depot, and dataset, we looked at the number of shared key driver genes between all other network pairs.The largest number of key drivers was shared between networks from the same dataset, which was significantly more than expected by chance (Figs S2A,B,E,and F and S3A,B,E,and F).When comparing networks from different datasets, the number of shared key drivers was only significantly more than random in some of the comparisons, and had much lower P-values (Figs S2C,D,G,and H and S3C,D,G,and H).  (Franz én et al, 2016), from subcutaneous and visceral adipose tissue were subsetted between males and females.Co-expression network genes and WHR adjBMI genomewide association study (Pulit et al, 2019) genes used to construct Bayesian networks representing each sex and depot using RIMBANET (Zhu et al, 2008).Key driver genes shared between the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task and Genotype-Tissue Expression project were identified in each network.53 key driver genes expressed in (pre-)adipocytes but unstudied in adipose tissue were prioritized for further study.Seven selected key driver genes identified were perturbed in human pre-adipocyte cells, and functional readouts of adipogenesis, Wnt signaling, proliferation, and mitochondrial oxygen consumption were collected.
Finally, we compared the identity of the key drivers in each network.In all comparisons, most key drivers were unique to one network.The most overlaps were found between networks from the same dataset, followed by overlaps between the STARNET and GTEx versions of the same network.More dissimilar comparisons generally had fewer shared key drivers, and few key driver genes were shared between three or four networks (Fig S4A -D).

Sample size differences partially explain differences in key driver prediction between male and female networks
We hypothesized that the increased predictive power of the male compared to the female networks could be because of the difference in input sample size.To test this, we randomly sub-sampled the male input genesets to include the same number of donors as the female networks, and constructed networks from these smaller sets.We identified an intermediate number of genes and edges for most sub-sampled networks (Table S7).There were also more shared key driver genes found in the sub-sampled male networks than between the full female networks.The predictions made by the sub-sampled networks recapitulated some of the full network predictions; both subcutaneous male sub-sampled networks (Table S7) recovered ANAPC2 and TYRO3 as key driver genes between the GTEx and STARNET versions.The smaller size and predictive power of the female networks appears to be only partially because of sample size differences.
Because we observed that accounting for the number of donors could only partially correct the differences in predicted key drivers, we hypothesized that something intrinsic to the female donors/ data may also contribute.We observe that the STARNET women are older than the GTEx women and may be entering menopause (Table 1).When comparing age to body fat distribution (WHR adjBMI ), we found that STARNET men had a significant positive correlation, whereas the STARNET women's age and WHR adjBMI were not significantly correlated (Fig S5A and B).
Finally, we know that transcription factor KLF14 is responsible for determining body fat distribution and related metabolic parameters in females (Small et al, 2018;Yang et al, 2022).We hypothesize that the differences in network structure may be because of regulation by KLF14.However, this gene was too lowly expressed in STARNET to be quantified and was not used to construct coexpression or Bayesian networks.In GTEx, the expression of KLF14 was significantly correlated with many of the shared key driver genes, although only a handful have correlations specific to females (Fig S6).Within the 334 shared key driver genes identified, only ACO1, ACAT1, and SLC25A1 are also part of the KLF14 transnetwork.
Prioritization pipeline identifies 53 novel and putatively functional adipocyte and pre-adipocyte key driver genes Because we hypothesize that fat distribution is driven, in large part, by pre-adipocyte and adipocyte expansion, we decided not to consider further any key driver genes that were primarily expressed in other cell types (see the Materials and Methods section, Fig 2A  and B).Most of the 110 removed genes were expressed primarily in immune cells (Fig S7).To use the strength of Bayesian networks in proposing unbiased hypotheses, we characterized key driver genes that were previously unstudied in adipocytes.We performed a comprehensive search of the existing literature to identify genes with known function in adipocytes.As a result, we removed 45 key driver genes, including FGF1, DPP4, LRP6, and RXRA, involved in adipocyte processes (Fig 2A and C, Supplemental Data 1).To identify genes that likely regulate adipocyte fat storage, we prioritized 41 key driver genes nearby WHR adjBMI GWAS loci and 15 key driver genes, when knocked out in mice, affect fat storage phenotypes (Fig 2A and D).In total, we prioritize 53 key driver genes with putative roles in human fat distribution via altered adipocyte fat storage that currently have unknown functions in adipose tissue (Table S6).Of the 45 well-studied key drivers, almost half were not near WHR adjBMI GWAS loci, nor did they affect fat storage in mice (Fig S8).The Bayesian network and key driver identification approach was to identify these functional genes without traditional evidence.

prioritized key driver genes have roles in Wnt signaling and mitochondria in other cell types
To further characterize the 53 key driver genes, we attempted to find enrichment of specific pathway genes, but did not identify any enriched gene ontology (GO) terms or msigDB Hallmark pathways, likely because of the removal of well-characterized genes (Fig S9A and B).We performed a second literature search to determine the primary function of the 53 genes in other cell types and found that 13 affect the activity of the Wnt signaling pathway in other cell types (Table 3, Supplemental Data 2).These 13 genes were identified as key driver genes in all four sex-depot networks, and 10 are WHR adjBMI GWAS candidate genes.We identified a different group of 13 genes that alter mitochondria function in other cell types, eight of which are WHR adjBMI GWAS candidate genes (Table 4, Supplemental Data 3).Many key driver genes were differentially expressed between depots, whereas fewer were differentially expressed between sexes (Fig S10A -D).
The expression of two key driver genes was significantly correlated with WHR adjBMI (Fig S11A -F).
Networks predict a role for four key driver genes in the Wnt signaling pathway Because canonical Wnt signaling can transcriptionally repress adipogenic genes PPARɣ and CEBPα (Teo & Kahn, 2010;Chen & Wang, 2018) (Fig 3A), we chose four of the 13 Wnt-related key driver genes that act upstream of β-catenin and TCF/LEF in other cell types.PSME3 (proteasome activator subunit 3, REG-ɣ, PA28-ɣ) (Li et al, 2015;Chen et al, 2017), RSPO1 (r-spondin 1) (Binnerts et al, 2007;Gore et al, 2011), and TYRO3 (TYRO3 protein tyrosine kinase) (Zhu et al, 2016;Chen et al, 2020) were previously shown to activate Wnt, while ANAPC2 (anaphase-promoting complex 2) inhibits Wnt signaling (Ganner et al, 2009).Their role in adipose remains unknown, although multiple lines of evidence suggest they may have a regulatory role in fat storage (Figs 3B-E, S12A-E, S13A-E, S14A and B, and S15A-E).In networks, these four genes regulate a large number of downstream genes (Figs 3B-E, S12C-E, S13E, S14B, and S15F), including WHR adjBMI GWAS candidate genes (Table S6), and canonical (A) 334 key driver genes prioritized to 53 putative candidate regulators of WHR adjBMI and adipocyte function using publicly available data.(B) Cell types in adipose tissue single cell and single nucleus RNA-seq data (Hepler et al, 2018;Min et al, 2019;Almanzar et al, 2020;Sun et al, 2020;Vijay et al, 2020;Hildreth et al, 2021;S árv ári et al, 2021) in which each key driver is expressed.(C) Key driver gene with known function in preadipocyte and adipocyte fat storage pathways.(D) Genetic evidence (status as WHR adjBMI genome-wide association study gene or causes mouse fat storage phenotype) for key driver genes.Data Information: The green checkmark indicates genes kept in the analysis pipeline, and the red X indicates genes removed.
Systems genetics analysis of body fat distribution genes Reed et al.
https://doi.org/10.26508/lsa.202402603vol 7 | no 7 | e202402603 Wnt signaling pathway genes.TYRO3's downstream genes are significantly enriched for lipid-related gene ontology (GO) terms (Fig S15D).ANAPC2, PSME3, and TYRO3 are located within the gene dense WHR adjBMI GWAS loci, and may be the causal gene at the locus.The signals near PSME3 and TYRO3 are both specific to females (Figs S13A-C and S15A-C).The lead single nucleotide polymorphism (SNP) in the ANAPC2 locus, rs144926207, is located in the intronic region of ANAPC2, and reference allele T is associated with higher WHR adjBMI (Pulit et al, 2019, Fig S12A) and higher excision of an intronic region of ANAPC2 in subcutaneous and visceral depots in GTEx (Fig S16A and B).RSPO1 shows strong differential expression between sexes and between depots (Fig S14A ).All four genes had strong correlations with WHR adjBMI in STARNET (Fig S11A -D) and these correlations differ by sex or by depot.By virtue of their status as prioritized network key driver genes, we hypothesize that these four genes affect fat storage in adipocytes, potentially through canonical Wnt signaling activity.

ANAPC2, PSME3, and RSPO1 overexpression alter adipogenesis, not proliferation
To test these hypotheses, we overexpressed each of the four Wnt key driver genes or a GFP control plasmid in a human male pre-adipocyte cell line (Wabitsch et al, 2001;Fischer-Posovszky et al, 2008) using lentivirus (see the Materials and Methods section, Fig 1).We confirmed the overexpression of each gene compared to GFP controls using qRT-PCR  (Fig 3F).We also overexpressed gene RSPO3 as a positive control because previous literature shows that RSPO3 does not affect subcutaneous pre-adipocyte proliferation but impairs adipogenesis (Loh et al, 2020).
We assessed the ability of these cell lines to proliferate by seeding each at the same density and counting representative wells every 24 h (see the Materials and Methods section).We found no differences in the rate of increase in cell number or the mean doubling time during exponential growth between any cell lines and GFP controls (Fig 3G and H).
We quantified the alterations in adipogenesis because of key driver overexpression by staining cells with the neutral lipid specific dye, Oil Red O (ORO), at 0, 6, and 12 d after the onset of differentiation.As expected, we observed an increase in lipid accumulation on day 6 and 12 compared with day 0 in all cell lines, confirming successful induction of adipogenesis, with a 4.25-fold increase in ORO absorbance in GFP controls on day 12 compared with day 0 (Fig 3I and J).Compared with GFP controls, ANAPC2 and RSPO1 overexpressing cells were deficient in adipogenesis, with 24.6% and 45.4% less lipid accumulation, respectively, than GFP controls on day 12.We observed that PSME3 overexpressing cells had a 31.6%increase in lipid accumulation compared with controls on day 12. TYRO3 overexpression caused no significant differences in lipid accumulation from controls at any time point.Positive control RSPO3 overexpressing cells were also deficient in lipid accumulation by 37.3% compared with controls on day 12, consistent with previous studies (Loh et al, 2020).Expression of adipocyte markers CEBPα, PPARɣ, and ADIPOQ also increased over 12 d, and showed significant inhibition in ANAPC2 and RSPO1 overexpressing cells compared with controls, in agreement with adipogenesis (Fig S17A -C).
To fully characterize the effects of these genes, we would need to perform the same functional studies in both subcutaneous and visceral cell lines to determine the magnitude of adipogenic alterations in each cell type and infer effects on body fat distribution.In the absence of these data, we used the correlations with WHR adjBMI in STARNET (Fig S11E) to determine if our data could explain the relationship between gene expression and body fat distribution.RSPO1 findings are well aligned with the human data-e.g., if RSPO1 inhibits adipocyte fat storage in both subcutaneous (Fig 3D) and visceral adipose tissue, then expression of RSPO1 in visceral adipose tissue should decrease visceral fat storage and hence decrease WHR adjBMI , and expression in subcutaneous adipose tissue should decrease subcutaneous fat storage to increase WHR adjBMI .This is perfectly replicated in STARNET correlation data (Fig S11E )-we observe positive correlations between subcutaneous RSPO1 expression and WHR adjBMI , but we observe negative correlations between visceral RSPO1 expression and WHR adjBMI .Although the correlation data do not immediately explain ANAPC2 and PSME3 findings, phenotypic effects in visceral adipocytes of different magnitudes or directions could explain the observed changes.

RSPO1 activates Wnt signaling to inhibit adipogenesis
We assessed the activity of the canonical Wnt signaling pathway using cells expressing both an overexpression plasmid and the 7TFC-luciferase construct, which measures the output of β-catenin-TCF/LEF transcription via luminescence (see the Materials and Methods section).PSME3 and RSPO1 overexpressing cells were able to activate canonical Wnt signaling significantly more than GFP controls; luminescence increased by 0.74-fold and 1.34-fold in PSME3 and RSPO1 overexpressing cells, respectively (Fig 4A).
Next, we looked at individual molecules of the canonical Wnt signaling pathway (Chen & Wang, 2018).We quantified the ratio of active (non-phosphorylated) β-catenin to total β-catenin using immunoblotting (Fig 4B and C), and we observed that PSME3 and RSPO1 overexpressing cells had a 25.4% and 33.1% increase in active/total β-catenin species compared with controls, respectively, consistent with the luciferase reporter assay.We quantified AXIN2 mRNA expression, a target of canonical Wnt signaling, using qRT-PCR (Fig 4D).We observed that, compared to GFP control cells, cells overexpressing ANAPC2 and RSPO1 increased AXIN2 expression by 1.9-fold and 2.2-fold, respectively.Finally, using ELISAs, we measured the ratio of active to total GSK3β, an inhibitor of the canonical Wnt signaling pathway (Fig 4E).Compared with GFP controls, we observed a 28.2% decrease in the ratio of active to total GSK3β in PSME3 overexpressing cells.
We also assessed the consequences of gene overexpression on non-canonical Wnt signaling (Ackers & Malgor, 2018;Akoumianakis et al, 2022).We observed a significant 21.7% decrease in the mRNA expression of IL6, a target of multiple types of non-canonical Wnt signaling, in PSME3 overexpressing cells (Fig 4F).RSPO1 overexpressing cells also show a non-significant 15.8% decrease in IL6 expression.Using ELISAs, we measured the ratio of active to total CAMK2A, a member of the Ca 2+ non-canonical Wnt pathway, and we observed a significant 70.2% and 85.0% decrease in active to total CAMK2A ratio in PSME3 and RSPO1 overexpressing cells, respectively (Fig 4G).Finally, we measured the ratio of active to total JNK, a member of the planar cell polarity non-canonical Wnt pathway.We observed no differences between overexpressing cell lines and GFP controls (Fig 4H ).Networks predict a role for four key driver genes related to mitochondrial function Because mitochondria can impact adipocyte function and lipid storage in various ways, we chose four of the 13 mitochondrialrelated key driver genes (Table 4) with diverse functions to study further.In other cell types, C1QTNF3 (Complement C1q Tumor Necrosis Factor-Related Protein 3) increases mitochondrial biogenesis, oxygen consumption, and ATP synthesis (Feng et al, 2016;Zhang et al, 2017;Gao et al, 2020), and MIGA1's protein (Mitoguardin 1) promotes mitochondrial fusion (Liu et al, 2016;Zhang et al, 2016).Psme3 is anti-apoptotic and promotes mitochondrial health in mice (Moncsek et al, 2015;Xie et al, 2020), and UBR1 (Ubiquitin Protein Ligase E3 Component N-Recognin 1) targets misfolded mitochondrial proteins (Tran, 2019;Metzger et al, 2020) and prevents mitophagy (Yamano & Youle, 2013).Their role in adipocytes remains unknown, although multiple lines of evidence suggest they may have a regulatory role in fat storage (Figs 5A-D, S13A-E, S18A-C, S19A and B, and S20A and B).In networks, these four genes regulate a large number of downstream genes (Figs 5A-D, S13E, S18C, S19B, and S20B), including WHR adjBMI GWAS candidate genes (Table S6) and genes related to mitochondrial function.We find that C1QTNF3 and PSME3 are found near WHR adjBMI GWAS loci along with other genes, and we hypothesize they may be the causal gene in these loci, whereas MIGA1 and UBR1 are putative mechanistic genes that are not in GWAS loci.Confirming their likely role in adipocyte mitochondria, we find that the expression of these genes is significantly correlated to UCP1 expression in adipose tissue (Fig S21).
Because these four genes are prioritized network key driver genes, we hypothesize that they affect mitochondrial function in adipocytes (Fig 1).

Knockdown of MIGA1 and UBR1 inhibits oxygen consumption in differentiated adipocytes
To test these hypotheses, we down-regulated each of the four genes using siRNA in primary human female pre-adipocyte cells, with nontargeting siRNA as a control (see the Materials and Methods section).We obtained mature adipocytes by differentiating these cells for 18 d, then measured the mRNA expression of each gene to confirm that the knockdown efficiency was still more than 50% compared to controls (Fig 5E).
We examined the effect of the knockdown of the remaining four genes on UCP1 expression in differentiated adipocytes.We found that C1QTNF3 and MIGA1 knockdown significantly reduced UCP1 expression, 71.1%, and 48.7%, respectively, compared with controls (Fig 5F).We also measured the expression of mature adipocyte markers, PPARG, CEPBA, and FAPB4.UBR1 knockdown resulted in a significant 40.6% increase in FABP4 expression (Fig S22A -C).
We then determined the effect of gene knockdown on cellular oxygen consumption rate (OCR) using the Seahorse assay (Ding et al, 2018) (see the Materials and Methods section).Importantly, we found that MIGA1 knockdown significantly reduced the basal OCR and OCR after adding ATP synthase inhibitor compared with controls (Fig 5G).In addition, we found that UBR1 knockdown significantly reduced the OCR after adding ATP synthase inhibitor compared with controls.Only MIGA1 knockdown resulted in significantly reduced extracellular acidification rate (ECAR) in differentiated adipocytes compared with controls (Fig 5H ).
Because each stimulation used in the OCR assay inhibits specific parts of the respiratory chain, we derived deeper mitochondrial phenotypes (Rose et al, 2014;Son et al, 2017)   oxygen consumed by mitochondrial proton leak, which refers to the futile H + shuttling across the outer mitochondrial membrane that does not produce ATP.Thermogenic uncoupling through UCP1 contributes to proton leak (Bertholet et al, 2022).Both MIGA1 and UBR1 knockdown cells showed significantly less proton leak compared with controls; proton leak was decreased by 40.4% and 39.9%, respectively (Fig 5J).

Discussion
We present the first large-scale investigation into gene regulators of body fat distribution in adipose tissue in a sex-and depotspecific manner.We constructed large Bayesian networks using male and female adipose tissue gene expression from subcutaneous and visceral samples, and we identified over 300 putative regulators spanning both sexes and depots.Using additional evidence, we prioritized 53 unstudied key driver genes that may affect adipocyte function and that putatively regulate body fat distribution.Because of the unbiased nature of our initial key driver selection, we were able to prioritize putative candidate GWAS genes, as well as putative causal genes that are not in GWAS loci.
In our study, we considered 495 genes nearby ~400 WHR adjBMI GWAS loci (Pulit et al, 2019) as candidate causal regulators of fat distribution.Other researchers have found that the nearest genes are likely, but not certainly, the mechanistic gene within the locus (Nasser et al, 2021), and problems identifying the causal gene at a locus often stem from multiple genes or signals (Franz én et al, 2016).Status as a replicated key driver provided independent, expression-based evidence that allowed us to prioritize 41 genes nearby WHR adjBMI GWAS loci as putatively mechanistic, including COL8A1, PSME3, and ANAPC2.We showed that PSME3 and ANAPC2 have novel functional effects on adipogenesis; COL8A1 was also prioritized in the colocalization studies by Raulerson et al (2019) and is likely a functional regulator of fat distribution as well.Of the 45 key driver genes already well characterized in adipocyte fat storage (Figs 2C and S8), 12 were nearby WHR adjBMI GWAS loci.By integrating gene expression and WHR adjBMI GWAS data, we can narrow the candidate causal gene list in an unbiased way.
Our network analyses were able to capture known biology, as well as make novel predictions.Most of the 45 well-studied key driver genes affected adipocyte fat storage by modulating adipogenesis, whereas a smaller number affected thermogenesis or lipolysis.Wellstudied key driver genes ACO1, ACAT1, and SLC25A1 are regulated in trans by KLF14; these genes may mediate KLF14's effects on female fat distribution.Because of the bulk nature of our adipose tissue gene expression datasets, we identified key drivers in other cell types and pathways that were not fully explored here.ARHGEF12 was identified as a key driver in all networks, making it an interesting candidate, although it was not prioritized here because it is not a candidate GWAS gene nor does it have strong effects in mice (Table S6).
Our analyses focused on 23 novel genes in two well-established pathways, Wnt signaling, and mitochondrial function, as putative drivers of adipocyte function; we demonstrated a functional role for five of the genes in these pathways.Two genes, PSME3 and RSPO1, showed antagonistic effects on canonical and Ca 2+ noncanonical Wnt signaling (Fig 4), consistent with previous studies (Akoumianakis et al, 2022).We showed that RSPO1 likely controls adipogenesis through its effects on Wnt signaling, consistent with the role of other r-spondins (Loh et al, 2020).RSPO1 is a novel serum marker of obesity (Kang et al, 2019) and may have a role in adipocyte beiging as well (Sun et al, 2023).However, Wnt signaling is unlikely the mechanism by which PSME3 controls adipogenesis, because of the directionally inconsistent effects.PSME3 is a member of the 20S proteasome (Funderburk et al, 2022) known to degrade GSK3β (Li et al, 2015), consistent with our results that show lower levels of active:total GSK3β in PSME3 overexpressing cells.Other 20S proteasome members affect fat storage (Kitamura et al, 2011;Willemsen et al, 2022), whereas PSME3 itself has diverse roles in many cell types (Dong et al, 2013;Funderburk et al, 2022;Lee et al, 2023).Although the implications in adipocytes warrant further study, non-canonical Wnt ligands cause inflammation and vascular disease (Akoumianakis et al, 2022) and are released from visceral fat more than subcutaneous fat (Fuster et al, 2015).ANAPC2 inhibited adipogenesis, although the loss of ANAPC2 overexpression on day 12 or lack of Wnt signaling changes may account for the small change in adipogenesis observed.ANAPC2 encodes part of the anaphase-promoting complex/cyclosome (APC/C) (Yu et al, 1998), which regulates the cell cycle, and others show that manipulating the expression of other APC/C proteins has consistent effects on adipogenesis (Lee et al, 2009;Du et al, 2022).Interestingly, we saw no differences in proliferation because of ANAPC2 overexpression (Fig 3).MIGA1 may be involved in thermogenesis because of its broad effects on mitochondrial function (Fig 5).Four validated key driver genes, MIGA1, ANAPC2, PSME3, and RSPO1, have a role in fertility and the development of gonads or gametes (Tomizuka et al, 2008;McGuinness et al, 2009;Huang et al, 2016;Liu et al, 2016;Geng et al, 2020), indicating a potentially sexspecific role.Three validated key driver genes, UBR1, ANAPC2, and PSME3, have known roles in protein degradation (Yu et al, 1998; (G) Oxygen consumption rates genes in NTC cells and in siRNA knockdown lines.(H) Extracellular acidification rates genes in NTC cells and in siRNA knockdown lines.(I) Phenotypes calculated from oxygen consumption rates under various stimulations.(J) Analysis of mitochondrial phenotypes upon siRNA perturbation under stimulations.Data Information: (A, B, C, D) Four selected key driver genes are regulated both WHR adjBMI downstream genes (Pulit et al, 2019) (yellow) and mitochondrial downstream genes (purple, GO term "Mitochondrion") in GTEx and Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task.(E, F, G, H, I, J) n = 6 replicates used in all assays.ROSI cells were treated with 2 μM rosiglitazone for 24 h before assay.(E, F, I) Differences between groups in (E, F, I) were determined using one-way ANOVA by gene (Gene of Interest versus NTC).(G, H) Differences between groups in (G, H) were determined using one-way ANOVA within each timepoint by gene (Gene of Interest versus NTC).All post hoc tests were performed using pooled t test with Dunnett's adjustment.Adjusted P-values shown with * (*** = adj.P < 0.001,* = adj.P < 0.05, # = adj.P < 0.1).Tran, 2019;Funderburk et al, 2022), which may be another important pathway regulating adipocyte function.
Fat distribution is a complex, full-body phenotype that is difficult to recapitulate in vitro and in vivo models.We used subcutaneous and visceral gene expression data to make predictions, but we tested those predictions on the cells available to us, a subcutaneous male pre-adipocyte cell line and primary subcutaneous pre-adipocytes from a female donor.To fully characterize the effects of these genes on adipocyte function and fat distribution, we need to repeat experiments in both sexes and compare these results with visceral cells.We hypothesize that some will have larger effect sizes on visceral cells or female cells.
The regulation and presentation of fat distribution differ between males and females, and similarly, the in silico gene regulatory structures captured by the networks were sex-specific.Of the 53 prioritized key drivers, only five genes are replicated key drivers in both male and female networks.Although it was unsurprising to find that the predicted key drivers were different, we found that the male networks could predict almost twofold more key driver genes than the female networks.For male networks, this was significantly more key drivers than expected by random chance (Supplemental Results).We found that the difference in sample size between males and females could partially explain the difference in predictive power.Neither age and apparent menopausal status, nor expression of female regulator gene KLF14 could explain the remaining differences in predictive power (Supplemental Results).
Our analysis compared two publicly available, independent datasets, STARNET and GTEx.There are large-scale differences between donors in these datasets, including living versus deceased donors, abdominal versus lower body subcutaneous adipose collection sites, cardiovascular disease status, age, ethnicity, BMI, and others (see the Materials and Methods section).These differences are reflected in the gene expression, which creates networks that are partially "overfit" to these samples, leading to the identification of key drivers that regulate dataset-specific processes.Accordingly, we find many key drivers shared between networks from the same dataset (Supplemental Results).By comparing the key drivers identified in STARNET and GTEx networks, we hope to filter out these datasetspecific key drivers, and identify genes that regulate the fat storage and homeostatic processes in the adipose tissue.Whereas our analysis successfully identified five genes with a role in adipocytes, some key drivers predicted by the networks were false positives and had no effect on adipogenesis or mitochondrial function.
We showed the validity and strength of Bayesian network modeling to predict known and novel gene regulators.We provided additional evidence of the role of Wnt signaling and mitochondria in adipocyte function and putatively in body fat distribution.Finally, we hypothesize a broader role for five genes in regulating fat distributions in humans.

Gene expression data
We interrogated RNA-sequencing gene expression data from subcutaneous adipose tissue and visceral abdominal adipose tissue from the GTEx (The Genotype-Tissue Expression, 2020) and the STARNET (Franz én et al, 2016).Detailed explanations of participant inclusion, data collection, sequencing, and quantification can be found at each source.Briefly, STARNET participants are people living with coronary artery disease, from whom biopsies of abdominal subcutaneous fat and abdominal visceral fat were obtained during open thorax surgery.Samples were sequenced using the Illumina HiSeq 2000 platform.GTEx biopsies of abdominal visceral fat and leg subcutaneous fat were taken from deceased donors shortly after death.Samples were sequenced using the Illumina TruSeq platform.Both datasets were obtained in transcripts per million format.

Expression data processing
We first used annotation meta-data from each source to divide the data into males and females.We used XIST expression to confirm these assignments.Next, we used annotations from the R package bioMart for genome build hg38 to select only the protein coding genes within each dataset.We then removed genes with less than 0.1 transcripts per million value in greater than 80% of the samples.Finally, we log transformed the gene expression values for subsequent analysis.

Bayesian network theory
Bayesian Networks are a type of Directed Acyclic Graph, where the relationships between the nodes in the graph are causal, or directed.Bayesian Networks have been used to determine directed connections between genes in an unbiased manner (Zhang et al, 2013(Zhang et al, , 2020;;M äkinen et al, 2014;Hamed et al, 2015;Huan et al, 2015;McKenzie et al, 2017;Shu et al, 2017Shu et al, , 2019;;Agrahari et al, 2018;Chella Krishnan et al, 2018;Carcamo-Orive et al, 2020;Hartman et al, 2021).Based on Bayes' theorem, the probability that a node in the network has a certain expression value depends on the expression of its parent nodes, or the nodes upstream of it.For gene A, with parent genes B and C, the probability that A has a particular expression value depends on the expression state of B and C: Each edge in the network represents one of these conditional relationships between genes.The total structure of the graph describes all relationships between genes as a joint probability.The joint probability of a full graph, X, is described as the geometric sum of all individual node probabilities.
where X is the full graph, X i is a node in the graph X, and D i is the set of parents for node X i .Popular methods to learn the relationships within the graph generally try to maximize a likelihood function, which increases the ability of the graph's joint probability to describe the observed gene expression data.Because multiple graph structures can result in the same likelihood, we also add prior information to improve predictions, such as known direct connections or eQTL data.
Bayesian network construction is a time and computationally intensive process, and few popular construction tools are able to handle networks with more than 100 nodes.The Reconstructing Integrative Molecular Bayesian Network (RIMBANET) (Zhu et al, 2004(Zhu et al, , 2008) ) tool is able to handle up to 10,000 nodes by discretizing the gene expression data to reduce computational complexity.RIMBANET also takes in diverse prior information, including direct connections, eQTL data, and continuous gene expression data, to improve the predictive power of the network.We chose RIMBANET to construct Bayesian networks for its ability to handle large input genesets and its reproducibility between datasets (Zhu et al, 2007;Cohain et al, 2017).

Bayesian network input genes
Ideally, we would probe gene-gene interactions at a genome-wide level, but Bayesian network construction is computationally intensive, and therefore, we limited this analysis to a subset of <10,000 genes that are more likely to regulate body fat distribution (Fig 1).We prioritized putative regulators of body fat distribution using three strategies: (1) genes whose expression are co-expressed with others in adipose tissue, (2) genes proximal to body fat distribution GWAS loci (Pulit et al, 2019), and (3) genes that are putatively regulated by the transcription factor KLF14 (Small et al, 2018).

Co-expressed genes
For a gene to be connected to others in a co-expression network, it must be expressed in the measured dataset, must vary between samples, and must be correlated with the expression of other genes.These properties are optimal for Bayesian network construction and can indicate gene function in the tissue of interest; therefore, we constructed adipose tissue co-expression networks for all eight datasets and identified genes connected to the corresponding STARNET and GTEx networks.We used the python package iterativeWGCNA (Greenfest-Allen et al, 2017 Preprint) to obtain modules of co-expressed genes in each dataset.Weighted gene co-expression network analysis (Langfelder & Horvath, 2008) uses correlations found within the data to determine which groups of genes are highly correlated and likely co-regulated.First, we computed the correlations between all genes.We raised these correlation coefficients to an empirically determined power to increase the differences observed.Next, we performed hierarchical clustering on the correlation matrix to define modules of highly correlated genes.We then assessed the success of this clustering, and iteratively reassigned genes to the modules in which they fit best.Lowly expressed or uncorrelated genes were not assigned a module.We identified which genes were assigned to modules in each of the eight datasets.We then compared the GTEx and STARNET module assignments for each depot and sex; we found genes assigned to modules in both datasets in the four depot and sex groups.We then took the union set of these four genesets as the co-expressed geneset, contained 7,928 genes and made up the bulk of the input to Bayesian network construction (Table S1).

KLF14 trans-eQTL network genes
We have previously demonstrated that KLF14 expression regulates fat distribution in both female mice and humans (Yang et al, 2022).SNP rs4731702 is significantly associated with KLF14 expression in cis in adipose tissue of multiple cohorts (Civelek et al, 2017;Small et al, 2018).The same variant is also associated with the expression of 385 genes across the genome in trans-(Table S1).We hypothesized that KLF14's effect on fat distribution is mediated by the genes it regulates, and we included 385 KLF14 putative target genes in the input geneset.

WHR adjBMI GWAS loci-adjacent genes
The largest WHR adjBMI GWAS meta-analysis to date was performed on primarily European ancestry and discovered 346 loci associated with WHR adjBMI (Pulit et al, 2019).Multiple sources have determined that the functional gene is the nearest gene to the locus in ~70% of cases (Nasser et al, 2021), so we identified 443 genes overlapping or nearest to the lead SNP (and SNPs with LD r 2 > 0.8) of 346 WHR adjBMI GWAS loci using haploReg (Ward & Kellis, 2016).Further, we used two studies that identified high-quality candidate genes using colocalization methods (Civelek et al, 2017;Raulerson et al, 2019), where the SNPs that affect association with WHR adjBMI also affect the expression of 59 candidate genes, which are more likely to be functional (Table S1).In total, we considered this combined set of 495 genes as WHR adjBMI GWAS genes in this study.Whereas this set does not contain all possible causal genes, it is likely enriched for them.
The union set of weighted gene co-expression network analysis module genes, KLF14 targets, and putative GWAS genes made up the input to Bayesian Network construction.For each dataset, the 8,492 gene expression values were discretized into "low" "medium" and "high" bins using k-means clustering.

Prior information
Because multiple graph structures can result in the same likelihood score, we can use prior information to improve confidence in the network structure.

eQTLs
After the central dogma, information flows from DNA to RNA; therefore, if SNPs influence the expression of gene A, gene A is more likely to be regulatory of other genes B and C, etc.For each dataset, we determined which genes had cis-eQTLs with SNPs < ±500 kb.These eGenes were more likely to be parent nodes in the Bayesian networks.Neither STARNET nor GTEx determined cis-eQTLs in a sexspecific manner, so these eQTL eGenes were nearly identical for male and female networks (Table S2).

Continuous data
Although the network is built on discretized gene expression data, RIMBANET is able to use continuous gene expression data to inform network construction.First, the continuous data are used to generate Pearson correlations between all genes.Correlations with significance P < 0.01 are used as prior information to determine possible parents and prioritize which edges to add or remove.

Bayesian network construction
Bayesian Networks for each dataset were constructed using RIM-BANET using the discretized gene expression data, a list of eQTL eGenes, and the continuous gene expression data.The RIMBANET shell script was adapted for implementation on the University of Virginia's high-performance computational cluster (Rivanna).RIMBANET was run with these tags: -C TRUE to specify continuous data, -w to add the continuous dataset, -d to add the discretized dataset, -e to add eQTL eGenes.RIMBANET creates 1,000 versions of each network with random initial seeds.RIMBANET uses a Markov blanket to identify potential parents for each gene, then iteratively adds edges with the highest prior information to create the best network structure to represent the data, as measured by the Bayesian Information Criterion (BIC).
where n is the number of nodes, k is the number of parameters estimated (edges), and L is the likelihood function.Maximizing this function results in the best fit graph structure that is not overly complex.Then, RIMBANET merges the 1,000 network versions by retaining edges present in 30% of the iterations.Finally, RIMBANET produces a directed acyclic graph by removing complete cycles.

Properties of biological networks
Biological networks share some common features, including reproducibility, scale-freeness, and small-worldness (Takahashi et al, 2012).Scale-free networks have degree distributions that follow the power law; most nodes have a small number of downstream genes, whereas a few nodes have a large number of downstream genes (Barabasi & Albert, 1999).The distribution of the number of downstream genes per parent can be fitted as a power law P(k) ~k−a , where k represents the number of downstream edges and P(k) represents the fraction of nodes that have k edges.The degree exponent, a, is determined by fitting the line log(P(k) ~−a log(k).In biological networks, the degree exponent is commonly 2-3.We used the igraph() package in R to calculate the degree exponent for each network.Small-world networks are highly clustered yet have a short average distance between nodes (Watts & Strogatz, 1998).We used the qgraph() package in R to calculate the clustering coefficient and average path length between nodes for each network.Because these properties scale with the number of nodes in the network, we compared these metrics with a random graph of the same size.

Key driver gene analysis
Key driver genes are genes that, because of their prominent position in the network, regulate many genes downstream.We were particularly interested in identifying key driver genes that regulate disease-related genes, specifically WHRadjBMI GWAS genes.We hypothesize that these in silico key driver genes also regulate disease-related gene programs in vitro.
Key driver genes of each network were identified with two methods: (1) by the number of downstream genes regulated by each potential key driver gene and (2) by the enrichment of disease genes in the set of downstream genes regulated by each potential key driver gene.
To identify type 1 key driver gene testing, first, every gene in the network was profiled to determine its number of downstream genes at distances 1-10 edges away using the shortest path.Then, the mean and SD in the number of downstream genes at each edge distance were calculated for the network.
For each potential key driver gene, we calculated 10 score functions: where n is the distance in edges, G is the number of downstream genes the potential key driver gene has at distance n, NM is the network mean number of downstream genes at distance n, NS is the network SD in the number of downstream genes at distance n.This is a metric of the extremeness of G, effectively a z-score.
Finally, we calculated a total score for each potential key driver gene by summing the "z-scores," weighting smaller edge distances away from the potential key driver higher than large edge distances: The top 10% the highest scoring genes in the network were declared type 1 key driver genes, similar to previous studies (Zhang et al, 2013).
To identify type 2 key driver genes, a "neighborhood" of downstream genes was declared for every gene in the network as those genes within four edges away.Next, WHR adjBMI GWAS genes (Pulit et al, 2019), defined above, were identified within the downstream neighborhood.Finally, using Fisher's exact test, we determined whether the number of GWAS genes in the downstream neighborhood was significantly more than expected by chance, compared with the whole network.Genes with significant downstream enrichments of WHR adjBMI GWAS genes were declared type 2 key driver genes.
Key driver genes for each network were the union set of type 1 and type 2 key driver genes.Shared key driver genes were genes identified as either type 1 or type 2 key driver genes in both STARNET and GTEx networks of the same type.

Testable key driver gene selection
Whereas this set of 334 replicated key driver genes likely contains many novel mechanistic drivers of fat distribution or fat storage, it also likely contains false positives and well-characterized genes.Furthermore, we hypothesize that fat distribution is driven, in part, by adipocyte expansion, and while genes involved in other adipose tissue processes, such as tissue structure, immune function, vascularization, etc., might contribute to fat distribution or its comorbidities, these were not the focus of this study.We used three steps to narrow this list to likely functional, testable adipocyte key driver genes.

Identification of cell type
We used seven publicly available single cell-or single nucleus-, adipose tissue-or adipose tissue-derived stromal vascular fraction RNA-sequencing datasets from both human and mouse (Hepler et al, 2018;Min et al, 2019;Almanzar et al, 2020;Sun et al, 2020;Vijay et al, 2020;Hildreth et al, 2021;S árv ári et al, 2021) to determine the primary cell type in which the gene was expressed.Because there was some disagreement between studies, all cell types in which the gene was expressed in any study are reported in Table S6.We removed genes that were only expressed in non-adipocyte cell types.

Identification of function in adipocytes
We used a comprehensive literature search to identify well-studied key driver genes (Supplemental Data 1).For each gene, we used GeneCards to identify alternate names for each gene or corresponding protein.We then searched PubMed and Google Scholar for functional studies in cells demonstrating a role for that gene in pre-adipocytes or adipocytes.Terms searched include "adipocyte," "adipogenesis," "differentiation," "lipogenesis," "lipolysis," "glucose uptake," "browning," and "thermogenesis."

Additional genetic evidence
We prioritized key driver genes based on two types of genetic evidence.First, we identified 41 WHR adjBMI GWAS genes (Pulit et al, 2019) within the set of remaining key driver genes.Their identity as a regulator of genes within the network and their location near a significant GWAS locus is strong evidence that they likely have a functional role in fat distribution.Second, we prioritized genes involved in fat storage and distribution in mouse models.We queried the Mouse Genome Informatics database (Blake et al, 2021) and the International Mouse Phenotyping Consortium (Groza et al, 2023) to determine if the gene knockout in mice results in significant differences in fat pad size, total body fat mass, lean mass, or related phenotypes.We did not consider overall body size differences, as these may be indicative of BMI-related phenotypes.Although there are differences in fat storage between mice and humans, there are many conserved pathways that point to shared genetic mechanisms and similar biological outcomes (Chusyd et al, 2016;B örgeson et al, 2022).We hypothesized that they play a similar role in human fat storage.

Lentivirus construction
Overexpression plasmids were constructed by VectorBuilder (VectorBuilder Inc) using the mammalian gene expression lentiviral vector backbone with one open reading frame.This backbone contains third generation lentiviral integration sites and ampicillin resistance.Using GTEx, we identified the most abundant isoform in adipose tissue for each gene.This isoform was added to the plasmid, followed by a P2A linker, then the GFP reporter sequence.This construct was under the CMV promoter.Control plasmids contain the GFP reporter gene under the CMV promoter with no P2A linker.
7TFC was a gift from Roel Nusse, purchased from Addgene (Addgene plasmid # 24307; Addgene).The 7TFC plasmid contains third generation lentivirus integration sites, the Firefly Luciferase gene under seven repeats of the TCF promoter, an mCherry marker under the SV40 promoter, and the ampicillin bacterial resistance gene.
We obtained the plasmids in E. coli swabs in agar.We cultured the E. coli on Luria-Bertani broth agar plates containing 100 μg/ml ampicillin and sub-cultured single colony-forming units in 50 ml Luria-Bertani broth containing 100 μg/ml ampicillin.Plasmids were isolated from E. coli using the Nucleobond Xtra Midi prep kit (Takara Bio) following the manufacturer's protocol (Cat# 740422.50).Plasmids were packaged into third generation replication-deficient lentivirus in HEK-293T cells using the Lenti-Pac HIV Expression Packaging Kit (Genecopoeia) following manufacturer's protocol (Cat# LT001).

Transduction and sorting of human pre-adipocyte overexpression cell lines
We obtained human male pre-adipocyte Simpson-Golabi-Behmel syndrome (SGBS) cells from Dr. Martin Wabitsch (Wabitsch et al, 2001) at passage number 35.There are no cell lines for female or visceral pre-adipocytes; only models using primary cells are available (Ruiz-Ojeda et al, 2016).All pre-adipocyte cells were grown and maintained as described previously (Fischer-Posovszky et al, 2008) in DMEM:F12 media (Thermo Fisher Scientific) containing 10% FBS, 1% Penicillin/Streptomycin, 8.1 ng/ml biotin and 3.5 ng/ml pantothenate.During transduction with lentivirus, the FBS was first heat-inactivated at 65°C for 30 min, and 8 μg/ml polybrene was added to improve transduction efficiency.We plated cells in 6-well plates and grew them to 70% confluence before transducing the cells with lentiviral particles containing each of the plasmids listed above.Cells containing high levels of GFP or mCherry were sorted using the FACS Aria Fusion Cell Sorter (BD Biosciences).We used cells with a passage number less than 46 for subsequent assays.

Transfection and differentiation of human primary pre-adipocytes
Human female subcutaneous primary pre-adipocytes were purchased from Zenbio (Cat# SP-F-SL; Lot# SL0061; Zenbio) and were differentiated according to the standard Zenbio white adipocyte differentiation protocol.Briefly, human primary subcutaneous preadipocytes were seeded on collagen-coated 96-well plate (20,000cell/well, 354650; Corning) with 200 μl of PM-1 medium (#PM-1; Zenbio), and the cells were established overnight, then transfected with siRNAs of target genes (Table 5) or scramble controls for 3 d using Lipofectamine RNAiMAX (cat# 13778-150; Invitrogen).The culture medium was replaced with 150 μl of differentiation medium DM-2 (#DM-2; Zenbio), and cells were cultured for 7 d.Media was replaced with maintenance medium AM-1, and cells were cultured for an additional 7 d for the following qRT-PCR and Seahorse experiments.

Quantification of gene expression in lentiviral treated human pre-adipocytes and differentiating cells
We grew cells in 12-well plates.Once they reached confluency, they were washed with PBS and incubated in 400 μl Trizol, then scraped and harvested.We extracted RNA using the RNeasy Micro Kit (QIAGEN), following the manufacturer's protocol (Cat# 74004).We digested the DNA species on the QIAGEN spin column using the RNAse-free DNAse kit (QIAGEN) following manufacturer's protocol (Cat# 79254).We quantified the isolated RNA using the Qubit with an RNA Broad Range assay kit (Thermo Fisher Scientific), following the manufacturer's protocol (Cat# Q10210).We reverse transcribed cDNA from the RNA templates using SuperScript IV Reverse Transcriptase Kit (Thermo Fisher Scientific) with Oligo(dT)20 primers, following the manufacturer's protocol (Cat# 18090010).We quantified cDNA abundance using qRT-PCR.Samples and standard curves were prepared using GoTaq qRT-PCR Master Mix (Promega) and gene specific primers (Integrated DNA Technologies) (Table 6).

Primer name Sequence
Primers used in adipogenesis/Wnt signaling assays Samples were measured using the QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific), and were analyzed using Thermo Fisher Scientific Connect qRT-PCR Standard Curve analysis software.

Quantification of gene expression in siRNA-treated human differentiated adipocytes
The total RNA samples were isolated from human differentiated adipocytes using KingFisher Flex Magnetic Particle Processor according to the MagMAX mirVana Total RNA isolation protocol.We obtained the cDNA samples from the RNA templates using SuperScript IV Reverse Transcriptase Kit (Thermo Fisher Scientific) with Oligo(dT)20 primers, following the manufacturer's protocol (Cat# 18090010).We quantified cDNA abundance using qRT-PCR using the QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific).The conditions were: 42°C for 5 min, a 10 s denaturation step at 95°C, followed by 40 cycles of 95°C for 5 s and 58°C for 40 s.

Proliferation assay
We plated 20,000 cells/well in 24-well plates.After 24 h, four wells were trypsinized, and cells were counted using a hemocytometer.Wells were washed with PBS and growth media was replaced.Every 24 h, four more wells were counted, for a total of 6 d.Growth media was replaced every 2 d.Most cells reached exponential growth by day 4 (Fig 4B).We then calculated the doubling time of each cell line using the formula: where T d is doubling time, T 1 and T 2 are initial and final time measurements, and N 1 and N 2 are the initial and final quantity of cells.

Quantification of adipogenesis
We quantified the amount of lipid stored in cells using Oil Red O (ORO) dye.0.25g of dye was suspended in 48 ml of 98% isopropanol and 32 ml of DI water.Unsuspended dye was removed from the ORO solution using 0.045 μM vacuum filtration.We repeated filtration (~3X) every 24 h until no precipitate was observed.Cells were washed with PBS, then fixed at 300 μl 4% PFA for 15 min.Cells were washed with 60% isopropanol, then dried completely.250 μl of ORO solution was added to the cells for 5 min.Cells were washed with DI water twice, then dried completely.We imaged the full wells using the EVOS microscope (below).ORO dye was then eluted from cells in 200 μl of 100% isopropanol for 2 min.Eluted ORO was quantified by measuring absorbance at 450 nm.

Imaging
We took images using the EVOS M7000 imaging system (Thermo Fisher Scientific) at 10x magnification, using phase contrast and color.We constructed full well composite images by taking 30 adjacent images in a 5 × 6 grid that covers most of the well.Composite images were stitched together using imageJ:Fiji plugin Grid/Collection Stitching (Schindelin et al, 2012).

Quantification of Wnt signaling transcriptional activation
We performed luciferase assays using the SGBS:7TFC reporter line.SGBS:7TFC cells were transduced with lenti-virus containing the gene of interest or GFP control plasmids.Images were taken to ensure a high percentage of dual mCherry and GFP-expressing cells.10,000 cells/well were plated in clear bottom, white-walled 96-well plates, with six replicates of each gene or control.After 24 h of incubation, luciferase activity was measured using the Luciferase Assay System (Promega) following manufacturer's protocol (Cat# E1500).Briefly, the cells were lysed in a 20 μl lysis buffer, 100 μl of luciferin-containing reagent was added, then emitted light was measured for 10 s using a luminescence plate reader.Luminescence readouts in each well were normalized to mCherry fluorescence to account for total luciferase insertions by the 7TFC cassette.

Quantification of OCR and ECAR
OCR and ECAR was determined using a Seahorse XF96 analyzer in combination with the Seahorse mitochondrial stress test kit according to a standard protocol (Ding et al, 2018).In brief, human primary pre-adipocytes were plated and differentiated as described above.Differentiated cells were washed with DPBS twice and incubated with Seahorse XF assay medium supplemented with 2 mM glutamax, 10 mM glucose, 1 mM sodium pyruvate (PH 7.4) for 45 min at 37°C in a non-CO2 environment.OCR and ECAR were subsequently measured in real time using XF96 extracellular flux analyzer (Seahorse Bioscience).The optimized concentration of compounds for mito-stress assay were 1.5 μM of oligomycin, 1.5 μM of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 μM of Rotenone and antimycin A. After the extracellular flux analysis, the OCR and ECAR were normalized by cell number, quantified using Hoechst staining.

Quantification of deep OCR phenotypes
We calculated basal mitochondrial respiration, ATP-linked respiration, proton leak, maximal respiratory capacity, reserve capacity, and non-mitochondrial respiration from the OCR assay as described previously (Rose et al, 2014;Son et al, 2017).We defined condition "A" as timepoints 1, 2, and 3 under basal stimulation; condition "B" as timepoints 4, 5, 6 under oligomycin stimulation; condition "C" as timepoints 7, 8, 9 under FCCP stimulation; and condition "D" as timepoints 10, 11, and 12 under Rot/AA stimulation.We considered each of the three timepoints within each condition as technical replicates, and the six samples as biological replicates.We averaged the three timepoints per condition into one value per biological replicate.We then defined non-mitochondrial respiration as D; basal mitochondrial respiration as A-D; ATP-linked respiration as A-B, proton leak as B-D; maximal respiratory capacity as C-D; and reserve capacity as C-A.

Statistical methods
Differences in proliferation and differentiation assays using the GFP-expressing control cells and cells expressing the gene of interest were assessed using two-way ANOVA by gene and time (day).Post hoc tests were performed between GFP controls and genes of interest within each timepoint using pooled t tests with P-value adjustment using Dunnett's adjustment.Differences in Wnt signaling using the GFP-expressing control cells and cells expressing the gene of interest and mitochondrial assays using the nontargeting control-expressing cells and cells with siRNA for the gene of interest were assessed using one-way ANOVA by gene.Post hoc tests were performed between controls and genes of interest using pooled t tests with P-value adjustment using Dunnett's adjustment.Analyses were performed using base R's anova() function and the emmeans package's emmeans() and contrasts().We reported only significant P-values, with the exception of

Figure 1 .
Figure 1.Overview of network construction, key driver gene identification, and functional validation.Publicly available RNA-seq gene expression data.The Genotype-Tissue Expression Project and Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (Franz én et al, 2016), from subcutaneous and visceral adipose tissue were subsetted between males and females.Co-expression network genes and WHR adjBMI genomewide association study(Pulit et al, 2019) genes used to construct Bayesian networks representing each sex and depot using RIMBANET(Zhu et al, 2008).Key driver genes shared between the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task and Genotype-Tissue Expression project were identified in each network.53 key driver genes expressed in (pre-)adipocytes but unstudied in adipose tissue were prioritized for further study.Seven selected key driver genes identified were perturbed in human pre-adipocyte cells, and functional readouts of adipogenesis, Wnt signaling, proliferation, and mitochondrial oxygen consumption were collected.

Figure 2 .
Figure 2. Prioritization of key driver genes for functional testing.(A)334 key driver genes prioritized to 53 putative candidate regulators of WHR adjBMI and adipocyte function using publicly available data.(B) Cell types in adipose tissue single cell and single nucleus RNA-seq data(Hepler et al, 2018;Min et al, 2019;Almanzar et al, 2020;Sun et al, 2020;Vijay et al, 2020;Hildreth et al, 2021;S árv ári et al, 2021) in which each key driver is expressed.(C) Key driver gene with known function in preadipocyte and adipocyte fat storage pathways.(D) Genetic evidence (status as WHR adjBMI genome-wide association study gene or causes mouse fat storage phenotype) for key driver genes.Data Information: The green checkmark indicates genes kept in the analysis pipeline, and the red X indicates genes removed.

Figure 3 .
Figure 3. Prioritized key driver genes RSPO1, PSME3, and ANAPC2 affect fat storage in adipocytes.(A) The Wnt signaling pathway consists of canonical β-catenin signaling and non-canonical pathways.13 key driver genes, shown in yellow and gray, interact with Wnt pathways in other cell types.(B) Key driver gene ANAPC2 in the Genotype-Tissue Expression project (GTEX) Subcutaneous Female network.(C) Key driver gene PSME3 in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task Visceral Female network.(D) Key driver gene RSPO1 in the GTEx Visceral Female network.(E) Key driver gene TYRO3 in the GTEx Subcutaneous Male network.(F) Expression of key driver genes compared with GFP controls on day 0 and 12 after onset of differentiation (n = 3).(G) Percent change in cell number over 6 d (n = 4).(H) Calculated doubling time in days (n = 12).(I, J) Quantification of Oil Red O staining of cells, performed for each gene of interest and GFP controls on day 0, 6 and 12 after beginning differentiation, (J) representative images of one well of a 12-well plate are shown(n = 3-15).Data Information: (B, C, D, E) Four selected key driver genes (red) regulate both WHR adjBMI downstream genes(Pulit et al, 2019) (yellow) and Wnt signaling downstream genes (blue, GO term "Wnt signaling pathway") in GTEx and Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task.(F, G, H, I, J) RSPO3 serves as a positive control.All plots show mean ± SEM.Differences between groups were determined using two-way ANOVA by day and gene (Gene of Interest versus GFP controls), and post hoc tests were performed using pooled t test with Dunnett's adjustment.Adjusted P-values shown with * (*** = adj.P < 0.001, ** = adj.P < 0.01, * = adj.P < 0.05).
(Fig 5I,  see the Materials and Methods section).For example, we calculated the amount of

Figure 5 .
Figure 5. Prioritized key driver genes MIGA1 and UBR1 affect mitochondrial function in adipocytes.(A) Key driver gene C1QTNF3 in the Genotype-Tissue Expression project (GTEx) Visceral Female network.(B) Key driver gene UBR1 in the GTEx Visceral Female network.(C) Key driver gene MIGA1 in the GTEx Visceral Male network.(D) Key driver gene PSME3 in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task Visceral Female network.(E) Gene expression of key driver genes in non-targeting control (NTC) cells and in siRNA knockdown lines.(F) Expression of UCP1 in siRNA knockdown lines and controls.(G)Oxygen consumption rates genes in NTC cells and in siRNA knockdown lines.(H) Extracellular acidification rates genes in NTC cells and in siRNA knockdown lines.(I) Phenotypes calculated from oxygen consumption rates under various stimulations.(J) Analysis of mitochondrial phenotypes upon siRNA perturbation under stimulations.Data Information: (A, B, C, D) Four selected key driver genes are regulated both WHR adjBMI downstream genes(Pulit et al, 2019) (yellow) and mitochondrial downstream genes (purple, GO term "Mitochondrion") in GTEx and Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task.(E, F, G, H, I, J) n = 6 replicates used in all assays.ROSI cells were treated with 2 μM rosiglitazone for 24 h before assay.(E, F, I) Differences between groups in (E, F, I) were determined using one-way ANOVA by gene (Gene of Interest versus NTC).(G, H) Differences between groups in (G, H) were determined using one-way ANOVA within each timepoint by gene (Gene of Interest versus NTC).All post hoc tests were performed using pooled t test with Dunnett's adjustment.Adjusted P-values shown with * (*** = adj.P < 0.001,* = adj.P < 0.05, # = adj.P < 0.1).
Fig 5 where indicated.All bar plots display the mean, with error bars displaying the SEM.

Table 3 .
Key driver genes that act in Wnt signaling in other cell types.

Table 4 .
Key drivers that act in mitochondria in other cell types.